(a) Field of the Invention
The present invention relates to genetically modified plant cells and plants wherein the genetic modification leads to the reduction of the activity of R1 and BEI and BEII proteins in comparison with corresponding plant cells of wild type plants that have not been genetically modified. Furthermore, the present invention relates to means and methods for the production thereof. Plant cells and plants of that type synthesise a modified starch characterised in that it has an amylose content of at least 75% and—in comparison with starch of corresponding wild type plants which have not been genetically modified—a reduced phosphate content and/or a modified distribution of the side chains and/or an increased gel strength in the texture analyser and/or a modified starch granule morphology and/or a modified average starch granule size. Thus, the present invention also relates to starch that can be synthesised by the plant cells and plants of the invention as well as methods for the production of this starch.
(b) Description of the Related Art
With regard to the increasing importance of plant ingredients as renewable raw material sources in the past few years, one of the problems of research in the field of biotechnology is to endeavour adjustment of these raw materials to the requirements of the processing industry. For allowing an application of renewable raw materials in as many as fields as possible, it is furthermore necessary to achieve a great variety of substances.
Apart from oils, fats and proteins, polysaccharides represent the essential renewable raw materials from plants. Among the polysaccharides, starch plays a central role beside cellulose. It is one of the most important storage substances in higher plants. For allowing as wide an application of starch as possible, it seems desirable to provide plants which are able to synthesise modified starch that is particularly suitable for different purposes. One possibility of providing such plants is—apart from cultivating—the purposeful genetic modification of the starch metabolism of starch-producing plants by genetic engineering.
The polysaccharide starch is a polymer of chemically uniform basic building blocks—the glucose molecules. It is, however, a very complex mixture of different molecule forms which differ with regard to their polymerisation degree and the occurrence of branchings of the glucose chains. Thus, starch is not a uniform raw material. There are two chemically different components of starch: the amylose and the amylopectin. In plants typically used for the production of starch, such as e.g. maize, wheat or potato, the synthesised starch consists of about 20%-30% of amylose starch and of about 70%-80% of amylopectin starch.
Amylose was considered a linear polymer for a long time, consisting of α-1,4-glycosidically bound α-D-glucose monomers. In recent studies, however, the presence of about 0.1% α-1,6-glycosidic branching points has been proven (Hizukuri and Takagi, Carbohydr. Res. 134, (1984), 1-10; Takeda et al., Carbohydr. Res. 132, (1984), 83-92).
As a rule, the complete separation of the amylose from the amylopectin is very difficult so that the quality of the amylose strongly depends on the type of the separation method chosen.
There are different methods for the determination of the amylose content. Some of these methods are based on the iodine-binding capacity of the amylose which can be determined potentiometrically (Banks & Greenwood, in W. Banks & C. T. Greenwood, Starch and its components (page 51-66), Edinburgh, Edinburgh University Press), amperometrically (Larson et al., Analytical Chemistry 25(5), (1953), 802-804) or spectrophotometrically (Morrison & Laignelet, J. Cereal Sc. 1, (1983), 9-20). The determination of the amylose content can also be carried out calorimetrically by means of DSC (differential scanning calorimetry) measurements (Kugimiya & Donovan, Journal of Food Science 46, (1981), 765-770; Sievert & Holm, Starch/Stärke 45 (4), (1993), 136-139). Moreover, it is possible to determine the amylose content by using the SEC (size exclusion chromatography) of native or debranched starch. This method was particularly recommended for the determination of the amylose content of genetically modified starches (Gérard et al., Carbohydrate Polymers 44, (2001), 19-27).
The choice of the analysis method used for the determination of the amylose content of a starch has a crucial influence on the size of the amylose figures determined as could be shown by various studies (Shi et al., J. Cereal Science 27, (1998), 289-299; Gérard et al., Carbohydrate Polymers 44, (2001), 19-27).
In contrast to the amylose, the amylopectin is branched to a larger degree and exhibits about 4% branching points which occur due to the presence of additional α-1,6-glycosidic linkings. The amylopectin is a complex mixture of glucose chains branched differently.
A further essential difference between amylose and amylopectin is the molecular weight. While amylose—depending on the origin of the starch—has a molecular weight of 5×105-106 Da, the molecular weight of amylopectin is between 107 and 108 Da. Both macromolecules can be differentiated from each other by their molecular weight and their different physico-chemical properties, which can be made apparent in the simplest way by their different iodine-binding properties.
The functional properties of the starch are strongly influenced—apart from the amylose/amylopectin ratio and the phosphate content—by the molecular weight, the pattern of the side chain distribution, the content of ions, the lipid and protein content, the average starch granule size and the starch granule morphology etc. Important functional properties to be mentioned are, for example, the solubility, the retrogradation behaviour, the water binding capacity, the film formation properties, the viscosity, the pasting properties, the freeze-thaw-stability, the acid stability, the gel strength etc. The starch granule size, too, can be important for different applications.
The ratio of amylopectin and amylose has a strong influence on the physico-chemical properties of the starches and, thus, on the possible applications of these starches. Since methods for the separation of these two components are very time-consuming and costly, such methods are no longer used on a large technical scale (Yound, A. H. in: Starch Chemistry and Technology. Eds. R. L. Whistler, J. N. BeMiller and E. F. Paschall. Academic Press, New York, 1984, 249-283). For a plurality of applications it would be desirable to have starches at disposal which still contain only one of the two polymers or at least one of the two starch components in an enriched form.
So far, both mutants and plants produced by genetic engineering have been described which, in comparison with corresponding wild type plants, exhibit a modified amylopectin/amylose ratio.
For example, the so-called “waxy” mutant from maize exhibiting a mutation in the gene encoding the starch granule bound starch synthase I (abbreviated: GBSSI) (Akasuka and Nelson, J. Biol. Chem., 241, (1966), 2280-2285; Shure et al., Cell 35 (1983), 225-233), produces a starch essentially consisting of amylopectin. For potato, genotypes were produced both by means of chemical mutagenesis of a haploid line (Hovenkamp-Hermelink et al., Theor. Appl. Genet., 225, (1987), 217-221) and by means of antisense inhibition of the GBSSI-gene, whose starches essentially consist of amylopectin starch. In comparison with starches of the corresponding wild type plants, such waxy potato starches do not exhibit any differences with regard to phophate content, the morphology of the starch granule or the ion content (Visser et al., Starch/Stärke, 49, (1997), 438-443).
Furthermore, maize mutants are commercially available which exhibit starches with amylose contents of about 50% or about 70% (amylose content determined by potentiometric determination of the iodine-binding capacity) and which are designated Hylon V® or HylonVII® (National Starch and Chemical Company, Bridgewater, N.J., USA). Moreover, also maize hybrids have been described which synthesise so-called “low amylopectin starch” (LAPS) and exhibit a content of high molecular (“high mol weight”) amylopectin of about 2.5% and an amylose content of about 90% (potentiometric determination of the iodine-binding capacity) (Shi et al., J. Cereal Science 27, (1998), 289-299).
Furthermore, transgenic potato plants have been described which, due to the antisense-inhibition of the branching enzyme I (=BEI) and the branching enzyme II (=BEII) gene, synthesise a potato starch which exhibits an amylose content of up to 75% by colorimetric determination of the amylose content according to the method described by Morrison and Laignelet (J. Cereal Sci. 1, (1983), 9-20) (Schwall et al., Nature Biotechn. 18, (2000), 551-554). These potato starches are characterised by a phosphate content of the starch which is up to 6 times higher compared to corresponding wild type plants. Furthermore, the international patent application WO 97/11188 describes transgenic potato plants which, due to their antisense inhibition of the R1 gene and the BEI gene synthesise a starch with an amylose content of more than 70%, the amylose content having been determined according to the method by Hovenkamp & Hermelink (Potato Research 31, (1988), 241-246).
Transgenic potato plant cells and potato plants synthesising a starch having an amylose content of more than 75% (calorimetric determination of the amylose content according to Hovenkamp & Hermelink (Potato Research 31, (1988), 241-246) and, at the same time, a reduced phosphate content in comparison with corresponding wild type plants have not been described in the state of the art so far. The same applies to the potato starches that can be isolated from these potato plant cells and plants and to methods for the production of such starches. However, the provision of such starches is desirable since their physico-chemical properties can be expected to be advantageously useful for various industrial applications.
Thus, the technical problem underlying the present invention is to provide plant cells and plants synthesising starch which has an amylose content of more than 75% (colorimetric determination of the amylose content according to Hovenkamp & Hermelink (Potato Research 31, (1988), 241-246) and a reduced phosphate content in comparison with the phosphate content of starch from corresponding wild type plant cells and plants that have not been genetically modified, as well as to provide such starch which differs from the starches described in the state of the art in its structural and/or functional properties and is, thus, more suitable for general and/or specific purposes.
This technical problem has been solved by providing the embodiments characterised in the claims.